Cross-Competition in Transgenic Chloroplasts Expressing Single Editing Sites Reveals SharedcisElements

Abstract

RNA editing in organelles of angiosperm plants results in alteration of Cs to Us in transcripts. In most editing sites analyzed in vitro or in vivo, sequences within approximately 30 nucleotides (nt) 5 and 10 nt 3 of the edited C have been found to be required for selection of the correct C editing target and for editing efficiency, but no consensus sequences have been identified. The effect of high-level expression of two different minigenes carrying either the rpoB-2 or the ndhF-2 editing site on editing was determined for all 31 known edited Cs in two transgenic tobacco lines. The editing efficiencies of both the corresponding rpoB and ndhF editing sites in the endogenous genes' transcripts and in several other genes' transcripts were reduced in the chloroplast transgenic plants. Conserved nucleotides could be identified in the sequences immediately 5 of each overexpressed editing site and in the sites in the additional genes that experienced a competition effect, though the conserved nucleotides differ 5 of rpoB-2 and 5 of ndhF-2. Inspection of sequences surrounding chloroplast and mitochondrial editing sites reveals that they can be grouped into clusters which carry conserved nucleotides within 30 nt 5 of the C target of editing. The data are consistent with a model in which the same trans factor recognizes several chloroplast or mitochondrial editing sites, depending on the particular sequence 5 of the edited C.Transcripts of vascular plant chloroplast and mitochondrial genomes are modified by C-to-U RNA editing (3,10,17,26,29). Angiosperm chloroplasts typically contain 25 to 31 known editing sites (28), while 441 sites have been detected in mitochondria of Arabidopsis, the only vascular plant with a mitochondrial genome that has been completely sequenced (11). RNA editing in angiosperm chloroplasts usually results in alterations of the second codon position in coding regions, changing predicted amino acids (28). Mitochondria exhibit predominantly coding-region editing, but editing in noncoding regions and silent third codon positions is also observed. In both chloroplasts and mitochondria, there is a preference for a 5Ј pyrimidine and 3Ј purine next to the C target of editing (2).The requirements for chloroplast transcript editing have been probed in chloroplasts by introducing transgenes carrying sequences surrounding editing sites. Such experiments have established that 16 to 40 nucleotides immediately 5Ј and 6 to 20 nucleotides 3Ј are critical for editing. The amount of surrounding sequence required varies somewhat depending on the particular editing site. While 21 nucleotides surrounding the psbL editing site allowed efficient editing, 84 nucleotides surrounding two ndhB editing sites did not result in any transgene transcript editing (4). Though mitochondrial transformation is not yet possible, similar conclusions can be drawn by examining naturally occurring mitochondrial genomes which have undergone chance recombination events that have placed extra, truncated fragments of editing genes into coding...

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…We have demonstrated that ORRM1 is a true editing factor because the effect cannot be explained as an indirect effect caused by changes in RNA transcript abundance. Reduced transcript abundance can indirectly affect the level of editing extent, if RNA is degraded before it is edited (31), or if transcript levels increase to levels high enough to saturate the editing machinery (32). We have verified that there is no significant difference in transcript abundance between the wild-type and the Arabidopsis orrm1 mutant by performing Northern blots that are consistent with the cpRNA-seq data (Table S1).…”

Cross-competition in Editing of Chloroplast RNA Transcripts in Vitro Implicates Sharing of Trans-factors between Different C Targets

“…Using gaps to align the rpoB C467 and rps14 C80 editing sites, as reported by Ref. 15, reveals six positions in common that negatively affect editing when mutated, and five of these six are the same nucleotide in both sequences (Fig. 4C).…”

“…The sequences surrounding all editing sites in a given organism do not show obvious similarity to each other either by direct sequence alignment or by secondary structure prediction. However, transplastomic tobacco that overexpress a fragment of maize rpoB or tobacco ndhF transcripts spanning the rpoB C467 or ndhF C290 editing sites, respectively, showed reduced editing at the cognate tobacco sites, as well as at additional sites (15), indicating that at least some ciselements are related. These three editing sites therefore form a "cluster" affected by overexpression of transcripts carrying only one C target (Fig.…”

“…Here we report our findings on the relationship of the cis-element of maize rpoB C467 to that of its cluster member, rps14 C80. The existence of a five-member rpoB C473 cluster has been functionally proven in tobacco using an in vivo approach (15); however, in the putative orthologous maize cluster, editing occurs only at rpoB C467 and rps14 C80 because the remaining three members of the maize cluster have a genomically encoded U at the position of the C target in tobacco (Fig. 1).…”

“…1). Furthermore, three 2-3-nt 2 regions of sequence identity exist between the sites of the rpoB C473 cluster within 20 nt 5Ј of the edited C, when gaps are introduced in the sequences (15).…”